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Sensitive and rapid diagnostic systems for avian influenza (AI) virus are required to screen large numbers of samples during a disease outbreak and to prevent the spread of infection. In this study, we employed a novel fluorescent dye for the rapid and sensitive recognition of AI virus. The styrylpyridine phosphor derivative was synthesized by adding allyl bromide as a stable linker and covalently immobilizing it on latex beads with antibodies generating the unique Red dye 53-based fluorescent probe. The performance of the innovative rapid fluorescent immnunochromatographic test (FICT) employing Red dye 53 in detecting the AI virus (A/H5N3) was 4-fold and 16-fold higher than that of Europium-based FICT and the rapid diagnostic test (RDT), respectively. In clinical studies, the presence of human nasopharyngeal specimens did not alter the performance of Red dye 53-linked FICT for the detection of H7N1 virus. Furthermore, in influenza A virus-infected human nasopharyngeal specimens, the sensitivity of the Red dye 53-based assay and RDT was 88.89% (8/9) and 55.56% (5/9) relative to rRT-PCR, respectively. The photostability of Red dye 53 was higher than that of fluorescein isothiocyanate (FITC), showing a stronger fluorescent signal persisting up to 8 min under UV. The Red dye 53 could therefore be a potential probe for rapid fluorescent diagnostic systems that can recognize AI virus in clinical specimens. [ABSTRACT FROM AUTHOR]

The circulation of the H9N2 virus results in significant economic losses in the poultry industry, and its zoonotic transmission highlights the need for a highly sensitive and rapid diagnostic and detection system for this virus. In this study, the performance of lateral flow test strips for a fluorescent immunochromatographic test (FICT) was optimized for the diagnosis of H9N2 virus-infected animal samples. The novel monoclonal antibodies (McAbs) against influenza A H9 viruses were developed, and two categories of McAbs with linear and conformational epitopes were compared for the performance of rapid diagnostic performance in the presence of feces sample at different time points (2, 4, and 6 days) post-infection (dpi). The limit of detection (LOD) of FICT and Kd values were comparable between linear and conformational epitope McAbs. However, superior performance of linear epitope McAbs pairs were confirmed by two animal studies, showing the better diagnostic performance showing 100% relative sensitivity in fecal samples at 6 dpi although it showed less than 80% sensitivity in early infection. Our results imply that the comparable performance of the linear epitope McAbs can potentially improve the diagnostic performance of FICT for H9N2 detection in feces samples. This highly sensitive rapid diagnostic method can be utilized in field studies of broiler poultry and wild birds. [ABSTRACT FROM AUTHOR]

Prevention is the most effective management strategy for influenza A infection in humans. In this study, we developed a CdSe/CdS/ZnS quantum dot (QD) fluorescent dye for rapid and sensitive detection of two common subtypes (H1N1 and H3N2) of influenza A virus, and examined its utility. CdSe/CdS/ZnS QD was conjugated with antibody (Ab) after conjugation with latex, making QD conjugate of QD + Latex + Ab. A stable photoluminescence of QD conjugate and advantage of CdSe/CdS/ZnS QD used was characterized in this study. The performance of a rapid fluorescent immunochromatographic test (FICT) employing QD conjugate (QD-FICT) in detecting influenza A/H1N1 was 8-fold and 64-fold higher than that of a europium nanoparticle-based FICT and a rapid diagnostic test (RDT; Standard Diagnostics BIOLINE Influenza A/B), respectively. For influenza A/H3N2, QD-FICT showed 8-fold and 128-fold higher performance than europium nanoparticle-based FICT and RDT, respectively. In clinical evaluations, QD-FICT showed 93.75% clinical sensitivity [45/48; 95% confidence interval (95% CI): 82.80–98.69], 100% clinical specificity (117/117; 95% CI: 96.90–100.00), and strong correlation (kappa; 0.98) with rRT-PCR (20 ≤ Ct ≤ 40). Europium nanoparticle-linked FICT showed 79.17% clinical sensitivity (38/48; 95% CI: 65.01–89.53) and 100% clinical specificity (117/117; 95% CI: 96.90–100.00), whereas RDT showed 77.08% sensitivity (37/48; 95% CI: 62.69–87.97), 100% specificity (117/117; 95% CI: 96.90–100.00), and reasonably good correlation with rRT-PCR (kappa; 0.93). Water-soluble QDs can therefore be used as an effective material for developing fluorescent diagnostic systems for rapid detection of human influenza A virus in clinical specimens. • CdSe/CdS/ZnS QD possessed higher quantum yield than (CuInS2) (CIS) QD. • Latex-mediated QD conjugate has better performance than polymer-coated QD conjugate to detect influenza A virus. • Photoluminescence (PL) of CdSe/CdS/ZnS quantum dot (QD) on latex was maintained in the presence of bovine serum albumin under UV irradiation. • QD-mediated FICT showed higher clinical sensitivity than europium nanoparticle by detecting the low amount of virus-infected patients. [ABSTRACT FROM AUTHOR]

Currently, the point of care testing (POCT) is not fully developed for subtype-specific avian influenza virus detection. In this study, an H5N1 hemaglutinin 1 (HA1) epitope (P0: KPNDAINF) and three modified peptides (P1: KPNTAINF, P2: KPNGAINF, P3: KPNDAINDAINF) were evaluated as POCT elements for rapid detection of avian influenza virus. Based on modeling predictions by Autodock Vina, binding affinity varied depending on alteration of one amino acid in these peptides. The binding energy of P2 indicated its potential for a strong interaction with HA. Fluorescence-linked immunosorbent assay experimentally demonstrated the interaction between these peptides and virus. The four peptides interacted with HA1 of H5N3 with different binding affinities with P2 showing the strongest binding affinity. When P0 and P2 peptides were used in rapid fluorescent immunochromatographic test (FICT) as detection elements, the inter-assay coefficients of variation (CV) indicated that P2-linked FICT was more acceptable than the P0-linked FICT in the presence of human specimens. Antibody pair-linked FICT was influenced by clinical samples more than the P2-linked FICT assay, which showed a 4-fold improvement in the detection limit of H5N3 and maintained H5 subtype-specificity. Compared to the rapid diagnostic test (RDT) which is not specific for influenza subtypes, P2-linked FICT could increase virus detection. In conclusion, results of this study suggest that HA epitope-derived peptides can be used as alternatives to antibodies for a rapid fluorescent diagnostic assay to detect avian influenza virus., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.